Neisseria gonorrhoeae, also called gonococci ("GC"), is the causative agent of gonorrhea. It is also one of the most prevalent sexually-transmitted bacterial pathogens worldwide; over 3 million cases are reported annually in the United States alone. GC invades and colonizes the mucosal surfaces of the urethra, cervix, rectum, throat and conjunctiva. GC is extremely sensitive to desiccation and temperature changes, and is almost exclusively transmitted by direct mucosal contact, such as during sexual intercourse. The sensitivity of the organism to drying and temperature changes has been problematic for diagnostic methods, particularly in less developed countries where specimens often need to be stored for long periods of time and/or transported to a diagnostic laboratory prior to analysis. Brock, BIOLOGY OF MICROORGANISMS 515-16 (3d ed., 1979); Jephcott, Genitourin. Med. 73, 245 (1997).
Gonorrhea remains a significant health problem worldwide, even in countries in which effective drug treatment is readily available. The symptomology of gonorrhea differs between infected males and females. In men, GC is most often manifested by a painful infection of the urethral canal. By contrast, in women, GC infection is often asymptomatic or causes only a mild vaginitis. Even in asymptomatic women, however, GC infection may result in pelvic inflammatory disease, infertility, and ectopic pregnancy. Asymptomatic female carriers may unintentionally spread the disease to their sexual partners and their newborns as the baby passes through the cervix and vaginal canal. Infants born to GC-infected women have an increased incidence of conjunctivitis and pneumonia. Iwen et al., J. Clin. Microbiol. 33, 2587 (1995). Early, rapid and inexpensive methods of identifying GC infection in potential carriers is essential to curbing the spread of this pathogen. Brock, BIOLOGY OF MICROORGANISMS 591-92 (3d ed., 1979); Crotchfelt et al., J. Clin. Microbiol. 35, 1536 (1997), Herrmann et al., J. Clin. Microbiol. 34, 2548(1996).
Conventional methods of identifying GC include gram staining, colony morphology, growth on selective media, and cytochrome oxidase testing. GC colonies are characterized as gram-negative, oxidase-positive, diplococci. Organisms from presumptively identified colonies of GC are frequently confirmed by sugar fermentation, fluorescent antibody staining, and/or coagglutination. Brock, BIOLOGY OF MICROORGANISMS (3d ed., 1979). However, such culture procedures are laborious, time consuming, and limited by the low viability of GC samples. Rapid and early identification of GC is desirable, especially in asymptomatic individuals, to slow the spread of the disease. In addition, because of the poor viability of stored GC specimens, diagnostic methods that avoid the culturing of viable organisms are advantageous.
To obviate the problems attendant to conventional diagnosis of GC, there have been attempts to develop nucleic acid based diagnostic methods for identifying GC.
Nucleic acid based diagnostic assays, such as Southern hybridization, offer rapid means of identifying microorganisms, usually in less than one day. Polymerase chain reaction (PCR)-based methods are even more sensitive and can sometimes provide results within hours. However, nucleic acid based methodologies are often fraught with drawbacks. Most of these methods are costly, are available for only a few species of microorganisms, and can resolve only one species per sample tested. Moreover, nucleic acid based assays require the development of oligonucleotide probes or primers that are specific for the microorganism of interest.
U.S. Pat. No. 5,536,638 to Rossau et al. teaches a method of identifying GC using a probe directed to a rRNA sequence. See also U.S. Pat. No. 5,432,271 to Barns et al., U.S. Pat. No. 5,389,515 to Chmelo et al., U.S. Pat. No. 5,378,606 to Stern et al., and U.S. Pat. No. 5,173,401 to Wolff et al. As many as 10,000 copies of the rRNA genes are present in bacteria; thus, diagnostic methods based on detection of the GC rRNA genes take advantage of this naturally-occurring amplification. American Society for Microbiology, DIAGNOSTIC MOLECULAR MICROBIOLOGY: PRINCIPLES AND APPLICATIONS (D. H. Persing et al., eds., 1993). In addition, the 16S or 23S rRNA genes are frequently used for probe development because variable regions exist within these highly conserved genes that can be used for species-specific detection. However, for certain organisms it may not be possible to derive highly specific and sensitive probes from the 16S and 23S rRNA genes, for instance, because their evolutionary nucleic acid sequence conservation is too high. Another consequence of the conserved character of these genes is that the differentiation of two organisms is often based on only one or a few mismatches in the target sequence, which puts constrains on the stringency of the hybridization. A slight deviation from these conditions might result in misidentification.
Totten et al., J. Infectious Diseases 148, 462 (1983), teaches a method of identifying GC in clinical specimens by detecting the "cryptic plasmid" commonly associated with GC. However, not all strains of GC contain the cryptic plasmid. The presence of this plasmid in different GC strains is highly variable, ranging from about 40% to about 96%, depending on geographic location. Thus, this identification method is of limited utility.
U.S. Pat. No. 5,256,536 to Miyada et al. teaches a method for detecting GC using a nucleic acid probe. The probe was identified after subtractive hybridization of GC DNA by N. meningitidis DNA.
U.S. Pat. No. 5,453,355 to Birkenmeyer et al. concerns oligonucleotide primers and probes for detecting GC by PCR amplification. The disclosed probes and PCR primers are directed to the pil E gene, which encodes the predominant surface antigen of GC.
Stary et al., J. Clin. Microbiol. 35, 239 (1997), discloses a method for identifying GC based on ligase chain reaction (LCR) amplification of a target sequence within the opa I gene. Buimer et al., J. Clin. Microbiol. 34, 2395 (1996), concerns a diagnostic test for simultaneously detecting both GC and Chlamydia trachomatis by LCR amplification using one set of primers directed against the opa genes of GC and a second set of primers targeting the C. trachomatis endogenous plasmid. Crotchfelt et al., J. Clin. Microbiol. 35, 1536 (1997) and Iwen et al., J. Clin. Microbiol. 33, 2587 (1995), provide assays for simultaneous detection of GC and C trachomatis by PCR coamplification and rRNA hybridization, respectively.
Notwithstanding the investigations described above, there remains a need in the art for rapid, accurate and sensitive methods for the identification of GC.